Department of Biochemistry and Biophysicshttp://hdl.handle.net/1957/19990

2015-03-03T18:53:12ZIncrease in cellular triacylglycerol content and emergence of large ER-associated lipid droplets in the absence of CDP-DG synthase functionhttp://hdl.handle.net/1957/55173
Increase in cellular triacylglycerol content and emergence of large ER-associated lipid droplets in the absence of CDP-DG synthase function
He, Yue; Yam, Candice; Pomraning, Kyle; Chin, Jacqueline S. R.; Yew, Joanne Y.; Freitag, Michael; Oliferenko, Snezhana
Excess fatty acids and sterols are stored as triacylglycerols and sterol esters in
specialized cellular organelles, called lipid droplets. Understanding what determines the cellular
amount of neutral lipids and their packaging into lipid droplets is of fundamental and
applied interest. Using two species of fission yeast, we show that cycling cells deficient in the
function of the ER-resident CDP-DG synthase Cds1 exhibit markedly increased triacylglycerol
content and assemble large lipid droplets closely associated with the ER membranes. We
demonstrate that these unusual structures recruit the triacylglycerol synthesis machinery and
grow by expansion rather than by fusion. Our results suggest that interfering with the CDPDG
route of phosphatidic acid utilization rewires cellular metabolism to adopt a triacylglycerol-rich lifestyle reliant on the Kennedy pathway.
This is the publisher’s final pdf. The published article is copyrighted by the author(s) and published by the American Society for Cell Biology. The published article can be found at: http://www.molbiolcell.org/.
2014-12-15T00:00:00ZAnalysis of clock-regulated genes in Neurospora reveals widespread posttranscriptional control of metabolic potentialhttp://hdl.handle.net/1957/54991
Analysis of clock-regulated genes in Neurospora reveals widespread posttranscriptional control of metabolic potential
Hurley, Jennifer M.; Dasgupta, Arko; Emerson, Jillian M.; Smith, Kristina M.; Freitag, Michael; et al.
Neurospora crassa has been for decades a principal model for filamentous
fungal genetics and physiology as well as for understanding
the mechanism of circadian clocks. Eukaryotic fungal and animal
clocks comprise transcription-translation-based feedback loops that
control rhythmic transcription of a substantial fraction of these transcriptomes,
yielding the changes in protein abundance that mediate
circadian regulation of physiology and metabolism: Understanding
circadian control of gene expression is key to understanding eukaryotic,
including fungal, physiology. Indeed, the isolation of clock-controlled
genes (ccgs) was pioneered in Neurospora where circadian
output begins with binding of the core circadian transcription factor
WCC to a subset of ccg promoters, including those of many transcription
factors. High temporal resolution (2-h) sampling over 48 h using
RNA sequencing (RNA-Seq) identified circadianly expressed genes in
Neurospora, revealing that from ∼10% to as much 40% of the transcriptome
can be expressed under circadian control. Functional classifications
of these genes revealed strong enrichment in pathways
involving metabolism, protein synthesis, and stress responses; in
broad terms, daytime metabolic potential favors catabolism, energy
production, and precursor assembly, whereas night activities favor
biosynthesis of cellular components and growth. Discriminative regular
expression motif elicitation (DREME) identified key promoter
motifs highly correlated with the temporal regulation of ccgs. Correlations
between ccg abundance from RNA-Seq, the degree of ccg-promoter
activation as reported by ccg-promoter-luciferase fusions, and
binding of WCC as measured by ChIP-Seq, are not strong. Therefore,
although circadian activation is critical to ccg rhythmicity, posttranscriptional
regulation plays a major role in determining rhythmicity
at the mRNA level.
To the best of our knowledge, one or more authors of this paper were federal employees when contributing to this work. This is the publisher’s final pdf. The published article is copyrighted by the National Academy of Sciences and can be found at: http://www.pnas.org/content/current.
2014-12-02T00:00:00ZA network of assembly factors is involved in remodeling rRNA elements during preribosome maturationhttp://hdl.handle.net/1957/54807
A network of assembly factors is involved in remodeling rRNA elements during preribosome maturation
Baßler, Jochen; Paternoga, Helge; Holdermann, Iris; Nyarko, Afua; Clark, Sarah A.; Barbar, Elisar; et al.
Eukaryotic ribosome biogenesis involves ~200 assembly
factors, but how these contribute to ribosome
maturation is poorly understood. Here, we
identify a network of factors on the nascent 60S subunit
that actively remodels preribosome structure. At its hub is
Rsa4, a direct substrate of the force-generating ATPase
Rea1. We show that Rsa4 is connected to the central
protuberance by binding to Rpl5 and to ribosomal RNA
(rRNA) helix 89 of the nascent peptidyl transferase center
(PTC) through Nsa2. Importantly, Nsa2 binds to helix 89
before relocation of helix 89 to the PTC. Structure-based
mutations of these factors reveal the functional importance
of their interactions for ribosome assembly. Thus,
Rsa4 is held tightly in the preribosome and can serve as
a “distribution box,” transmitting remodeling energy from
Rea1 into the developing ribosome. We suggest that a
relay-like factor network coupled to a mechano-enzyme is
strategically positioned to relocate rRNA elements during
ribosome maturation.
This is the publisher’s final pdf. The published article is copyrighted by the author(s) and published by the Rockefeller University Press. The published article can be found at: http://jcb.rupress.org/.
2014-11-17T00:00:00ZGleaning Unexpected Fruits from Hard-Won Synthetases: Probing Principles of Permissivity in Non-canonical Amino Acid–tRNA Synthetaseshttp://hdl.handle.net/1957/54535
Gleaning Unexpected Fruits from Hard-Won Synthetases: Probing Principles of Permissivity in Non-canonical Amino Acid–tRNA Synthetases
Cooley, Richard B.; Karplus, P. Andrew; Mehl, Ryan A.
The site-specific incorporation of non-canonical amino acids (ncAAs) into proteins is an important tool for understanding biological function. Traditionally, each new ncAA targeted requires a resource-consuming process of generating new ncAA aminoacyl tRNA synthetase/tRNACUA pairs. However, the discovery that some tRNA synthetases are “permissive,” in that they can incorporate multiple ncAAs, means it is no longer always necessary to develop a new synthetase for each newly desired ncAA. Developing a better understanding of what factors make ncAA-synthetases more permissive would increase the utility of this new approach. Here, we characterize two synthetases selected for the same ncAA that have markedly different “permissivity profiles.” Remarkably, the more permissive synthetase even incorporates an ncAA for which we had not been able to generate a synthetase using de novo selections. Crystal structures reveal that the two synthetases recognize their parent ncAA through a conserved core of interactions, with the more permissive synthetase displaying a greater degree of flexibility in its interaction geometries. We also observe that intra-protein interactions not directly involved in ncAA binding can play a crucial role in synthetase permissivity and suggest that designing such interactions may provide an avenue to engineering synthetases with enhanced permissivity.
This is an author's peer-reviewed final manuscript, as accepted by the publisher. The published article is copyrighted by John Wiley & Sons, Inc. and can be found at: http://onlinelibrary.wiley.com/journal/10.1002/%28ISSN%291439-7633
2014-08-18T00:00:00Z